4.2 PROVEEDORES
4.2.1 Diferencia Entre Proveedor Y Acreedor
The membrane internalisation inhibitors, chloroquine, ammonium chloride and bafilomycin Aj were used to investigate the effect o f inhibiting internalisation during a wt HSV 17^ infection o f DRG neurones. Chloroquine and ammonium chloride are lipophilic, acidotropic weak bases and in their unprotonated form enter cells and endosomes readily. Upon entering an acidic environment they become protonated and too polar to pass back through the endosome membrane. The fast change (minutes) in endosomal pH is due to neutralisation o f protons by the weak base.
Ba^ilnomycin specifically inhibits vacuolar type H^ ATPases (V-ATPase) which are responsible for the acidification o f vacuolar compartments such as clatherin coated pits, endosomes and lysosomes. Inhibition o f the V-ATPases results in neutralisation o f acid endosomes. The change in pH inhibits endosomal enzymes which inhibits internalisation by preventing the dynamic cycling between endosomes and the plasma membrane.
It is worth noting that wt HSV 17^ does not depend on endocytosis and acid pH for their penetration in contrast to Influenza virus and Semliki Forest virus (White et al.
1981; M organ et al. 1968). Inhibitors o f endocytosis which neutralise acid endosomes should not affect wt HSV 17^ adsorption or penetration. Just in case, however, the internalisation inhibitors were added to the medium 1 hour after infection with wt HSV 17^ to allow time for viral adsorption and penetration.
To determine the effects o f inhibiting internalisation on the sodium currents o f DRG neurones infected with wt HSV 1 7 \ lOOjiM chloroquine, 25mM ammonium chloride and Iq M bafilomycin A] were separately added to the media o f infected and uninfected DRG neurone cultures which were then incubated for a further 24 hours. Sodium currents were generated from a holding potential o f -80mV by a step depolarisation to +10mV for 13 msec (this protocol will be referred to as the one step depolarisation protocol throughout this chapter). Representative sodium current traces are shown in figure 5.1a. Figure 5.1b shows the sodium current amplitudes normalised for cell size (nA/pF) o f wt HSV 1Ÿ infected DRG neurones and infected or uninfected DRG neurones treated with internalisation inhibitors. The normalised sodium current amplitudes o f wt HSV 17^ infected DRG neurones were significantly smaller than those o f infected or uninfected DRG neurones treated with internalisation inhibitors. Infected and uninfected DRG neurones treated with internalisation inhibitors did not vary significantly in normalised sodium current amplitude. Additionally, the wt HSV 17^ infected DRG neurone treated with the internalisation inhibitors all had sodium currents. This demonstrates that inhibitors o f internalisation were able to prevent the loss o f sodium conductance which was observed after wt HSV 17^ infection for 24 hours.
bafflorrçdnAl bafilonydnAl andIBV m v
J
control BafA, chloro
0.00 control HSV control HSV control HSV control HSV
\ -0.05 -
%
I -0 .1 0 -11
1 3-0.15-1 -0.2 0- n=16 n=107T
n=20 n=20T
n=23 n=16 n=10 n=13F igu re 5.1. T he effect o f internalisation inhibitors on control and w t H S V I?"*" infected
D R G n eurones, a Sodium current traces of infected and unifected DRG neurones treated in the
presence or absence of bafilomycin A l. b The mean normalised current amplitude ± s.e.m of control and wt HSV 17+ infected DRG neurones treated with inhibitors o f internalisation, bafilomycin A \ (IpM ), chloroquine (lOOpM), and ammonium chloride (25mM). The only significant difference was between control current amplitudes and wt HSV 17+ infected current amplitudes, P<0.0001. The current amplitudes of wt HSV 17+ infected DRG neurones were significantly different to the current amplitudes of DRG neurones treated with bafilomycin A j, chloroquine or ammonium chloride during wt HSV 17+ infection for 24 hours, P<0.0001. The normalised sodium current amplitudes of DRG neurones treated with internalisation inhibitors were not significantly different from each other, P>0.5. Statistical analysis was examined by the Kruskal-Wallis statistical test, followed by the Kolmogorov-Smirnov pairwise comparison.
The sodium current-voltage relationships were constructed to investigate the possibility that inhibitors o f internalisation had an effect on the voltage dependence o f the sodium currents which could not be identified by analysis o f the currents generated after a depolarisation to a single voltage. Prior to evoking sodium currents a prepulse to -120mV was applied to remove sodium channel inactivation. The family o f sodium currents was then evoked by a series o f command potentials at 5mV intervals between -50mV and +25mV. The peak of each current evoked was plotted against command potential to construct a current-voltage relationship. The data were normalised with respect to the maximum current evoked for each neurone. The mean normalised current-voltage relationships are shown in figure 5.2 for uninfected DRG neurones treated with inhibitors of internalisation. Families o f sodium currents described throughout this chapter were generated with the same voltage protocol described above. The sodium current-voltage relationships which appear within this chapter were also constructed as described above. The family o f sodium currents were generated after a prepulse to -120mV to remove sodium channel inactivation. The current-voltage relationships show that a prepulse to -120mV did not reveal a current in neurones where no current had been seen with the one step depolarisation protocol.
-60 -20
potential (mV) -0.2-
-0.4-
-0.6-
Figure 5.2. The effect o f treatm ent with internalisation inhibitors on the current-
voltage relationship o f DRG neurones. The graph shows the normalised current
amplitudes ± s.e.m from control ( • ) n=5 DRG neurones, and bafilomycin A]
(A)
n=5, chloroquine(V)
n=5, and ammonium chloride ( □ ) n=7 treated DRG neurones for 24 hours.The activation curves were also constructed for each cell tested by normalising the conductance with respect to peak conductance (g/gmax). The mean normalised conductance-voltage relationships are shown in figure 5.3. DRG neurones incubated in the presence o f ammonium chloride or bafilomycin Aj show almost overlapping activation curves as reflected in the mean slope factors and V5 0 values shown in table
5.1 (page 175). The mean activation curve o f DRG neurones incubated in the presence o f chloroquine appears to be slightly shifted from the others. The error bars, however, are large and the mean slope factors and V5 0 values were not significantly different
from the others, (see table 5.1, page 175). There was no shift in the activation curves sufficient to affect the normalised sodium amplitude at +10 mV. Thus incubating DRG neurones in the presence o f internalisation inhibitors did not significantly alter the voltage dependence o f sodium currents.
0.6- g/gmax 0 . 4 - 0.2- -5 0 -4 0 -3 0) -20 potential (mV) - 1 0
Figure 5.3. The effect o f treatment with internalisation inhibitors on the
conductance-voltage relationship o f DRG neurones. The graph shows the normalised conductance ± s.e.m from control ( • ) n=5, and from DRG neurones whilst being treated with, bafilom ycin A)
(A) n=5, chloroquine ( V ) n=5, and ammonium chloride ( □ ) n=7.
The continuous lines were obtained by fitting the mean normalised data to a Boltzmann function.The normalised current-voltage relationships for wt HSV 1Ÿ infected DRG neurones treated with inhibitors of internalisation were calculated as described above for uninfected DRG neurones and are shown in figure 5.4. Incubation of wt HSV 17^ infected DRG neurones for 24 hours with inhibitors of internalisation did not cause a shift in mean normalised sodium current-voltage relationships.
-20
g -0.2 - ■ o
- 0 . 4 -
-0.6 = _
Figure 5.4. The effect o f wt HSV 17+ infection o f DRG neurones in the presence o f internalisation inhibitors on the current-voltage relationship. The graph shows the normalised current amplitudes ± s.e.m from control ( • ) n=5, and from wt HSV 17+ infected DRG neurones whilst being treated with, bafilomycin A]
(A)
n=9, chloroquine (▼ ) n=3, and ammonium chloride (■ ) n=5.For each family of sodium currents generated the conductance was calculated and the normalised conductances (g/gmax) were plotted against command potential, (figure 5.5). The activation curves of the wt HSV 17^ infected DRG neurones treated with internalisation inhibitors overlay each other and there is no significant difference in the mean slope factor and V50 values. This data is displayed in table 5.1, page 175.
0.6- g/gmax 0 . 4 - 0.2- -5 0 -4 0 -3 0 -2 0 potentail (mV) - 1 0
Figure 5.5. The effect o f wt HSV 17"^ infection o f DRG neurones in the presence o f internalisation inhibitors. The graph shows the norm alised conductance ± s.e.m from control ( • ) n=5, and from wt HSV I?"*" infected DRG neurones whilst being treated with, bafilomycin A]
(A)
n=9, chloroquine (▼ ) n=3, and ammonium chloride (■ ) n=5. The continuous lines were obtained by fitting the mean normalised data to a Botlzmann function.The loss of sodium conductance which is usually associated with a wt HSV 17^ infection of DRG neurones was not observed when the infected cells were incubated in the presence of chemicals which inhibit the internalisation of membrane proteins. The presence of internalisation inhibitors did not alter the normalised sodium current amplitude or the voltage dependence of the sodium currents evoked from infected or uninfected DRG neurones.
The pH of the media containing the internalisation inhibitors was measured. The media containing chloroquine changed in pH from pH 7.4 to pH 7.0 (a 3 fold increase in H^ concentration). The other internalisation inhibitors ammonium chloride and bafilomycin Aj, did not alter the pH of the media.